Weak O2 Binding and Strong H2O2 Binding at the Non-Heme Diiron Center of Trypanosome Alternative Oxidase.

Alternative oxidase (AOX) catalyzes the four-electron reduction of dioxygen to water as an additional terminal oxidase, and the catalytic reaction is critical for the parasite to survive in its bloodstream form. Recently, the Xray crystal structure of trypanosome alternative oxidase (TAO) complexed with ferulenol was reported and the molecular structure of the non-heme diiron center was determined. The binding of O-2 was a unique side-on type compared to other iron proteins. In order to characterize the O-2 binding state of TAO, the O-2 binding states were searched at a quantum mechanics/molecular mechanics (QM/MM) theoretical level in the present study. We found that the most stable O-2 binding state is the end-on type, and the binding states of the side-on type are higher in energy. Based on the binding energies and electronic structure analyses, O-2 binds very weakly to the TAO iron center (Delta E =6.7 kcal mol(-1)) in the electronic state of Fe(II)...O = O, not in the suggested charge transferred state such as the superoxide state (Fe(III)-O-O center dot-) as seen in hemerythrin. Coordination of other ligands such as water, Cl-, CN-, CO, N-3(-) and H2O2 was also examined, and H2O2 was found to bind most strongly to the Fe(II) site by.E = 14.0 kcal mol(-1). This was confirmed experimentally through the measurement of ubiquinol oxidase activity of TAO and Cryptosporidium parvum AOX which was found to be inhibited by H2O2 in a dose-dependent and reversible manner.